Conventional methods of purifying viruses and viral nucleic acids have significant faults and shortcomings. Conventional protocols for purifying bacterial and mammalian viruses from host cells or growth media generally contain three steps. First, viruses must be liberated from the host cells. Viruses which lyse the infected cells used to propagate the virus of course are released directly into the growth medium. However, certain other non-enveloped viruses, such as reovirus and adenovirus, are associated with membrane components of the cells and must first be extracted away from this material. A common method of extracting non-enveloped viruses from cellular components is by homogenizing the cell suspension in the presence of Freon 113 as described in Shatkin, A. J., Proc. Natl. Acad. Sci USA 54 1721 (1965). Although effective, care must be taken in performing this procedure, since it produces virus-containing aerosols and releases Freon into the environment.
Second, the virus must be concentrated prior to actual purification. Two methods are commonly used to concentrate and partially purify viruses. Viruses may be precipitated by addition of ammonium sulfate or polyethylene glycol, as discussed in Mahy B. W. J., Ed. Virology, a practical approach, Washington D.C. (1985). However, this method usually co-precipitates many other proteins present in the tissue culture medium, and therefore may to some extent reduce the purity of the virus sample. Alternatively, concentration of virus may be achieved by pelleting viruses in a sample via ultracentrifugation, leaving many soluble proteins in the supernatant, followed by the redissolving viruses in a small amount of buffer.
Third, the concentrated virus must be purified from extraneous materials. This last step in the purification process usually is performed by some form of fractionation employing density gradient ultracentrifugation. For example, the purification of reoviruses involves sedimentation banding on sucrose gradients followed by density equilibrium banding on cesium chloride, as described in Smith, R. E. et al., Virology 39 791 (1969), while picornaviruses have traditionally been purified by use of one of the two banding techniques, described in Rueckert, R. R. and M. Pallansch, (1981) Methods in Enzymology 78 315-326).
These conventional methods of isolating viruses from biological fluids, aqueous suspensions or solutions comprising biological fluids, require either exceedingly long times or expensive equipment for the centrifugation; and further require expensive equipment and/or use of toxic chemicals.
One approach to improving virus-isolating techniques has been to selectively adsorb viruses onto a solid material. An ideal adsorbent would selectively adsorb virus under certain conditions from extraneous materials in liquid suspensions, and desorb viable viruses under different condition to permit physical separation of viral particles.
Various synthetic polymeric materials have been employed in this approach. The cross-linked water soluble polymers of U.S. Pat. Nos. 3,224,941 and 3,684,777 are said to absorb water and adsorb, or inactivate, viruses. The water-soluble polymeric materials of U.S. Pat. No. 4,271,028 are said to adsorb viruses across the pH range of 5-10.
Synthetic polymeric materials which are water insoluble have also been employed in attempts to adsorb viruses. Most of these however have been pH insensitive, so that desorption of viruses would not occur upon change of pH. The materials of Johnson et al., Nature 665-667 (1967) were said to be useful for adsorbing viruses from highly dilute aqueous liquids, while the materials of U.S. Pat. No. 4,421,653 were said to adsorb proteins, including viruses.
Certain investigators have employed synthetic water-insoluble polymeric materials to adsorb viruses at acidic pH and to desorb them at elevated pH. However, these materials have generally been used to treat only very high volumes of water intended for drinking. Wallis et al., Applied Microbiology, 1007-1014 (1969); Wallis and Melnick, Water Research, 4 787-796 (1970); Wallis et al., Applied Microbiology, 703-709 (1971); and Wallis et al., Applied Microbiology, 740-744 (1972). Materials used to remove viruses from smaller volumes of aqueous material, such as the polymeric materials disclosed in U.S. Pat. No. 3,398,092, are said to remove or inactivate virus present in water.
LambdaSorb.RTM., from the Promega Corporation (Madison, Wis.) is a further solid material said to be useful in removing virus particles (in particular, bacteriophage lambda particles) from aqueous suspensions. This material is a conjugate of fixed Staphylococcus aureus cells and rabbit polyclonal antibodies directed against bacteriophage lambda particles. The adsorbent is shaken with a bacterial cell lysate, then centrifuged at 12,000.times.g for less than one hour to remove the adsorbent and any bound bacterial virus. An aqueous suspension of the adsorbent is used in a volume ratio of adsorbent to lysate of 1:100. Apparently, the bacteriophage do not desorb from the LambdaSorb, for only disruption of bound bacteriophage particles is disclosed.
Synthetic polymeric materials said to be useful in removing protein from aqueous suspensions are described in U.S. Pat. No. 5,294,681 and U.S. patent application Ser. No. 08/207,274, filed Mar. 7, 1994 by Krupey. These materials are water insoluble polycarboxylic acid compositions. They are added to and mixed with a suspension containing proteins for approximately 15 minutes to allow for formation of a polymer-protein matrix. Depending on the composition's substitution groups, the pH of the suspension is from pH 3 to 7.5. After the matrix is removed from the suspension, it is said the bound proteins may be released therefrom by washing the matrix in buffer solutions at pH 8.6 to 9.5, optionally in the presence of 0.5 to 2% w/w surfactant per volume of matrix pellet.
The techniques for purifying viral nucleic acids generally comprise the conventional virus purification steps enumerated above, followed by disruption of the viral particle with compounds such as surfactants or guanidine thiocyanate, centrifugation to partially purify the nucleic acid material, and electrophoresis. The drawbacks of conventional methods for purifying viruses--long time periods or the use of expensive equipment, and the use of toxic chemical substances--are also encountered in methods for purifying viral nucleic acids.
The nucleic acids purified from mammalian viruses using these conventional methods are fairly pure, since the viruses released into extracellular medium are largely separable from host cells and their components. However, nucleic acids isolated from bacterial viruses using these methods are often not as pure. The electrophoresis gels of conventionally purified bacteriophage nucleic acids typically do not reveal a single sharp band indicative of purity, but instead exhibit an elongated faint smear with several bands, indicating the presence of nucleic acids of many disparate lengths, including molecules longer than the known length of the viral nucleic acid molecule.
A common method for isolating the nucleic acid of bacteriophage lambda is disclosed in Molecular Cloning, A Laboratory Manual, 2nd Ed., Sambrook et al., pages 2.73-2.81, Cold Spring Harbor Laboratory Press, (1989). In this method, bacteriophage particles isolated using centifugation techniques are disrupted by exposure to heat, detergent (as a protein denaturant) a chelating agent, such as EDTA. The nucleic acid released by this disruption is isolated and purified from other viral components by phenol/chloroform extraction. Despite the presence of protein denaturants in this method, purified bacteriophage nucleic acid usually contains low levels of bacteriophage exonuclease, which cleaves the viral nucleic acid into many small pieces. An ongoing problem with conventional techniques of bacteriophage nucleic acid isolation is that despite use of heat and chemical denaturants, viral exonucleases and nucleic acid-binding proteins may retain bioactivity, and thus cleave or bind to the released nucleic acid respectively, thus greatly reducing the usefulness of the nucleic acid. Thus, subsequent extraction steps to remove proteins and other extraneous viral material from viral nucleic acid are frequently necessary.
Moreover, where nucleic acid is to be isolated from bacterial viruses adsorbed to a solid material, the use of heat and detergent may denature the protein so much that large amounts of viral protein is desorbed from the solid material by disruption, causing the resulting nucleic acid to contain substantial amounts of viral protein.
LambdaSorb.RTM., described above, is also said to be useful in isolating nucleic acid from bacteriophage. Heat in the presence of EDTA is applied in order to disrupt bacteriophage bound to the solid; the nucleic acid released by the disrupted bacteriophage is then isolated using phenol/chloroform extraction.